Construction And Function Evaluation Of The Bioactive Coating Of Fusion Protein VE-cadherin/Mfp-5

Cardiovascular implant materials are closely related to human diseases treatment and quality of life,whereas the persistent patency of vascular stent remains to be improved.Cardiovascular implant materials,such as vascular stents,directly contact with blood and wall tissue in vivo,and damage the implant site by mechanical strain.Therefore,the restenosis and thrombosis caused by slow endothelialization are the main problems in clinical,and the main reasons of these problems are the surface properties of implant material.So there are higher requirements for the surface properties of biological materials in clinical application.It is great significance that apply surface modification to make the cardiovascular plants have multifunction,such as acceleration of endothelialization,non-thrombogenesis,anti-inflammatory and inhibition of intimal hyperplasia.VE-cadherin,an endothelial specific adhesion molecule located at junctions between endothelial cells,constitutes the adheren junction of endothelial cells by the interaction of extracellular domain and plays an important role in angiogenesis and maintaining of vascular barrier.Mfp-5 is a mussel adhesive protein with most Dopa with good biocompatibility and can adhere tightly to diverse substrates under water.In this study,we constructed a fusion protein VE-M,VE-cadherin extracellular domain EC1-2 fused with Mfp-5,as the novel bioactive coating to effectively accelerate the endothelialization of stent through using the specific interaction of VE-cadherin extracellular domain as well as strong adhesion of Mfp-5.VE-M was applied to modify the surface of 316 L stainless steel(316L SS),and then the functions of fusion protein were proven through a series of assays,including characterization of coating,blood compatibility test,adhesion assay of endothelial cells and endothelial progenitor cells,detection of endothelial function on various coatings and implantation of stent in rats.The main research and conclusions are as follows:1.Construction and expression of fusion protein VE-M.The cDNA sequence of VE-cadherin extracellular domain EC1-2 was fused with the cDNA sequence of Mfp-5,and was cloned in the expression vector PET32a to generate PET32a(+)-VE-M,which was transformed into E.coli(DE3)for induction of VE-M fusion protein.Then VE-M fusion protein purification was performed by immobilizedmetal affinity chromatography,and VE-M was confirmed by SDS-PAGE analysis and western blotting.2.Preparation and characterization of VE-M coating.The VE-M fusion protein coating was prepared through dip-coating method based on 316L SS.Fluorescence labeled VE-M was found to form uniform protein layers under fluorescence microscope.The kinetics of coating formation was traced by SPR,which proved that VE-M has strong adhesive ability to substrate surface.The results of AFM(Ra=3.442 nm±0.73nm)and water contact angle(58.9°±4.0°)was obtained.This result showed that VE-M coating has strong adhesive ability to substrate with possible good blood compatibility and biocompatibility.3.Blood compatibility of VE-M coating.Hemolysis test showed that the hemolysis rate of VE-M coating was 1.195±0.470%,which is far less than 5%(the reference standard of biological material).In platelet adhesion assay,the number of adherent platelets was significantly reduced and these platelets did not extend pseudopodia or aggregate on VE-M coating.The result proved that VE-M fusion protein has good blood compatibility.4.The effect of VE-M on endothelial cells and endothelial progenitor cell adhesion.The result of cell adhesion experiment showed that the number of endothelial cells and endothelial progenitor cells on VE-M was 5.5-and 1.8-fold of that on bare 316L SS,respectively,and also was significantly more than that on other coatings.The neutralization test demonstrated that VE-M promotes endothelial cells adhesion due to the interaction of VE-cadherin extracellular domain.Meanwhile,we found that the expression of endogenous VE-cadherin was higher on VE-M coating,which further confirmed the positive impact of VE-M.This result indicated that VE-M effectively enhances the adhesion of endothelial cells and EPCs by the specifically interaction of VE-cadherin extracellular domain.5.Effect of VE-M on endothelial proliferation and function.The test of cell proliferation activity showed that the Ki67~+cells increased significantly on VE-M coating.Then we also proved that the junction of endothelial cells was tighter on VE-M than that on other subatrate by fluorescence staining of cytoskeleton,permeability test and quantitative PCR.The expression of eNOS was found to be increased greatly by ELISA.In addition,the number of monolayer endothelial cells adhered on VE-M coating was not increased significantly.This result demonstrated that VE-M contributes to the proliferation and junction of endothelial cells but does not enhance the risk of endothelial inflammation.6.The animal experiments of vascular stent coated by VE-M.The immunofluorescent staining of in-stent en face after stent implantation showed that VE-M coating captures more EPCs and forms large endothelium with better morphology and function than bare stent.HE staining proved VE-M coating can promote endothelium regrowth with little hyperplasia and good biological safety.SEM results suggested that repaired endothelium on VE-M coating stent had better function.This result demonstrated VE-M can accelerate endothelialization.